Isolation of the [3H]gabapentin-binding protein/alpha 2 delta Ca2+ channel subunit from porcine brain: development of a radioligand binding assay for alpha 2 delta subunits using [3H]leucine.
The novel antiepileptic agent gabapentin (Neurontin) binds with high affinity to the alpha 2 delta subunit of a voltage-dependent Ca2+ channel. We report here a simple purification scheme for detergent-solubilized alpha 2 delta subunits from porcine brain. This involves sequential chromatography on Q-Sepharose, Cu(2+)-charged iminodiacetic acid-Sepharose, wheat germ lectin-agarose, and Mono Q. The purified protein was essentially homogeneous by SDS-polyacrylamide gel electrophoresis with a subunit Mr of 145,000. Using [3H] gabapentin as the radiolabeled tracer and (S)-3-isobutyl gamma-aminobutyric acid to define nonspecific binding, the overall purification factor was 2760-fold and the apparent yield 26.6%. We also developed and validated a novel binding assay for alpha 2 delta Ca2+ channel subunits using the ligand pair L-[3H]leucine/L-isoleucine. Even in binding assays of crude brain membrane fractions, [3H]leucine proved to be remarkably stable and specific for the alpha 2 delta Ca2+ channel subunit. [3H]Leucine offers several advantages over custom-labeled [3H]gabapentin: it has a higher specific activity, is relatively inexpensive, and is available from commercial sources.
Brown
J P
JP
Parke-Davis Neuroscience Research Centre, Cambridge University Forvie Site, Cambridge, United Kingdom.
Dissanayake
V U
VU
Briggs
A R
AR
Milic
M R
MR
Gee
N S
NS
D000085:Acetates / Q000378:metabolism*; D000588:Amines; D000818:Animals; D015220:Calcium Channels / Q000302:isolation & purification* / Q000378:metabolism; D002540:Cerebral Cortex / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003509:Cyclohexanecarboxylic Acids; D000077206:Gabapentin; D007700:Kinetics; D007930:Leucine / Q000378:metabolism*; D046911:Macromolecular Substances; D009474:Neurons / Q000737:chemistry; D011485:Protein Binding; D011869:Radioligand Assay; D013552:Swine; D014316:Tritium; D005680:gamma-Aminobutyric Acid
D000085:Acetates; D000588:Amines; D015220:Calcium Channels; D003509:Cyclohexanecarboxylic Acids; D046911:Macromolecular Substances; D014316:Tritium; D005680:gamma-Aminobutyric Acid; D000077206:Gabapentin; D007930:Leucine